Kimberly Holloway - Academia.edu (original) (raw)

Papers by Kimberly Holloway

Frontiers in Immunology

IntroductionWe present here a strategy to identify immunogenic neoantigen candidates from unique ... more IntroductionWe present here a strategy to identify immunogenic neoantigen candidates from unique amino acid sequences at the junctions of fusion proteins which can serve as targets in the development of tumor vaccines for the treatment of breastcancer.MethodWe mined the sequence reads of breast tumor tissue that are usually discarded as discordant paired-end reads and discovered cancer specific fusion transcripts using tissue from cancer free controls as reference. Binding affinity predictions of novel peptide sequences crossing the fusion junction were analyzed by the MHC Class I binding predictor, MHCnuggets. CD8+ T cell responses against the 15 peptides were assessed through in vitro Enzyme Linked Immunospot (ELISpot).ResultsWe uncovered 20 novel fusion transcripts from 75 breast tumors of 3 subtypes: TNBC, HER2+, and HR+. Of these, the NSFP1-LRRC37A2 fusion transcript was selected for further study. The 3833 bp chimeric RNA predicted by the consensus fusion junction sequence is ...

Neuron Signaling in Metabolic Regulation

Cancer Research

Cancer proteogenomics combines genomics, transcriptomics and mass spectrometry-based proteomics t... more Cancer proteogenomics combines genomics, transcriptomics and mass spectrometry-based proteomics to gain insights into cancer biology and treatment responsiveness. While proteogenomics analyses have already shown great potential to deepen our understanding of cancer tissue complexity and signaling, how a patient’s tumor changes upon treatment has largely been the province of genomics. This is due to technical difficulties associated with doing proteogenomic analysis on clinic-derived core-needle biopsies. To address this critical need, we have developed a “microscaled” proteogenomics approach for tumor-rich OCT-embedded core needle biopsies. Tissue-sparing specimen processing (“Biopsy Trifecta EXTraction”, BioTExt) and microscaled proteomics (MiProt) methodologies allowed generation of deep-scale proteogenomics datasets, with copy number and transcript information for >20,000 genes and mass spectrometry-based identification and quantification of nearly all expressed proteins in a ...

Biology Open

Hepatoblastoma (HB) is the most common pediatric primary liver malignancy, and survival for high-... more Hepatoblastoma (HB) is the most common pediatric primary liver malignancy, and survival for high-risk disease approaches 50%. Mouse models of HB fail to recapitulate hallmarks of high-risk disease. The aim of this work was to generate murine models that show high-risk features including multifocal tumors, vascular invasion, metastasis, and circulating tumor cells (CTCs). HepT1 cells were injected into the livers or tail veins of mice, and tumor growth was monitored with magnetic resonance and bioluminescent imaging. Blood was analyzed with fluorescence-activated cell sorting to identify CTCs. Intra- and extra-hepatic tumor samples were harvested for immunohistochemistry and RNA and DNA sequencing. Cell lines were grown from tumor samples and profiled with RNA sequencing. With intrahepatic injection of HepT1 cells, 100% of animals grew liver tumors and showed vascular invasion, metastasis, and CTCs. Mutation profiling revealed genetic alterations in seven cancer-related genes, while ...

$Research paper thumbnail of Abstract 2997: Novel orthotopic patient-derived xenograft mouse models of hepatoblastoma that replicate tumor heterogeneity, chemoresistance, and refractory disease$

Cancer Research, 2021

Introduction: Hepatoblastoma (HB) is the most common pediatric primary liver tumor and has the fa... more Introduction: Hepatoblastoma (HB) is the most common pediatric primary liver tumor and has the fastest rising incidence of all pediatric solid tumors. Patients with high-risk, treatment refractory, or relapse disease have a survival rate of less than 50%. The development of clinically relevant models of these aggressive tumors will facilitate studies to identify drugs that target these cells. Methods: Fresh, whole primary tumor samples were implanted into the livers of immunocompromised mice. Tumor growth was monitored with MRI and ELISA to measure serum human Alpha-fetoprotein (AFP), which is detectable in the blood of tumor-bearing animals. Tumors were validated with immunohistochemistry (IHC) for HB markers Glypican-3 (GPC3) and Beta-catenin; short tandem repeat (STR) DNA validation; next generation sequencing-based mutation profiling of 124 genes involved in pediatric solid tumors; and RNA sequencing (RNA-seq). Cells derived from tumors were grown in vitro and used for high thro...

Cancer proteogenomics integrates genomics, transcriptomics and mass spectrometry (MS)-based prote... more Cancer proteogenomics integrates genomics, transcriptomics and mass spectrometry (MS)-based proteomics to gain insights into cancer biology and treatment efficacy. A proteogenomics approach was therefore developed for frozen core biopsies using tissue-sparing specimen processing with a “microscaled” proteomics workflow. For technical proof-of-principle, biopsies from ERBB2 positive breast cancers before and 48-72 hours after the first dose of neoadjuvant trastuzumab-based chemotherapy were analyzed. ERBB2 protein and phosphosite levels, as well as mTOR target phosphosites, were significantly more suppressed upon treatment in cases associated with pathological complete response, suggesting MS-based pharmacodynamics is achievable. Furthermore, integrated analyses indicated potential causes of treatment resistance including the absence of ERBB2 amplification (false-ERBB2 positive) and insufficient ERBB2 activity for therapeutic sensitivity despite ERBB2 amplification (pseudo-ERBB2 posi...

Molecular and Cellular Biology / Genetics, 2019

Emerging evidence suggests that ESR1 fusion transcripts that produce functional fusion proteins i... more Emerging evidence suggests that ESR1 fusion transcripts that produce functional fusion proteins in estrogen receptor positive (ER+) breast cancer play a role in acquired endocrine therapy resistance. We recently reported two in-frame ESR1 fusion transcripts, ESR1-YAP1 and ESR1-PCDH11X, identified in patients with endocrine therapy resistant breast cancer. Both ESR1 fusions generated stable in-frame, fusion proteins that were transcriptionally active, driving endocrine therapy resistant proliferation and also promoting an epithelial-to-mesenchymal transition (EMT) transcriptional program leading to metastasis in experimental models. Here, we extend our studies by examining properties of additional ESR1 fusions with diverse partner genes, ESR1-DAB2, ESR1-GYG1, ESR1-SOX9, ESR1-ARNT2, ESR1-PCMT1, and ESR1-ARID1B, all identified in endocrine-refractory breast tumors, to better understand the role of ESR1fusion gene formation in endocrine therapy resistance. Structurally, ESR1-ARNT2, ESR1-PCMT1, and ESR1-ARID1B fusions retain the first 6 exons of ESR1, therefore lacking ESR1 exons encoding the ligand-binding domain (LBD) that endocrine therapies recognize, but are instead fused in-frame to C-terminal sequences from partner genes, which follows the same fusion pattern of previously identified ESR1-YAP1, ESR1-PCDH11X, ESR1-DAB2, ESR1-GYG1, and ESR1-SOX9 fusions. ESR1-ARNT2, ESR1-PCMT1, and ESR1-ARID1B constructs, along with ESR1-DAB2, ESR1-GYG1, and ESR1-SOX9 constructs were stably expressed in an ER+ breast cancer cell line, T47D, and all produced stable fusion proteins. ESR1-ARNT2 and ESR1-SOX9 promoted hormone-independent and fulvestrant-resistant cell proliferation that was sensitive to cyclin-dependent kinase (CDK) 4/6 inhibitors, palbociclib and abemaciclib. In addition, ESR1-ARNT2 induced hormone-independent activation of estrogen responsive genes (TFF1, GREB1, and PGR), and EMT genes (SNAI1 and VCAN) along with upregulation of Snail protein. These results indicate that ESR1-ARNT2 and ESR1-SOX9 have similar functional and pharmacological properties to our previously described ESR1-YAP1 and ESR1-PCDH11X fusions. These data further support a role for ESR1 fusions in driving not only endocrine therapy resistance but also activating the metastatic process. Ongoing studies are examining the ability of ESR1 fusions to induce EMT phenotypes and to determine structure function relationships of ESR1 fusions. Although the formation of ESR1 fusions likely confers resistance to all endocrine therapies that target the LBD, ESR1 fusion driven growth remained sensitive to CDK4/6 inhibitor treatment therefore providing rationale for testing ESR1 fusion detection in guiding treatment with CDK4/6 monotherapy. Citation Format: Jonathan T. Lei, Xuxu Gou, Sinem Seker, Vaishnaivi Devorakonda, Kimberly R. Holloway, Adrian V. Lee, Dan R. Robinson, Matthew J. Ellis. Functional significance of ESR1 fusions with diverse gene partners in endocrine therapy resistant breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3479.

Cell reports, Jan 7, 2018

RNA sequencing (RNA-seq) detects estrogen receptor alpha gene (ESR1) fusion transcripts in estrog... more RNA sequencing (RNA-seq) detects estrogen receptor alpha gene (ESR1) fusion transcripts in estrogen receptor-positive (ER) breast cancer, but their role in disease pathogenesis remains unclear. We examined multiple ESR1 fusions and found that two, both identified in advanced endocrine treatment-resistant disease, encoded stable and functional fusion proteins. In both examples, ESR1-e6>YAP1 and ESR1-e6>PCDH11X, ESR1 exons 1-6 were fused in frame to C-terminal sequences from the partner gene. Functional properties include estrogen-independent growth, constitutive expression of ER target genes, and anti-estrogen resistance. Both fusions activate a metastasis-associated transcriptional program, induce cellular motility, and promote the development of lung metastasis. ESR1-e6>YAP1- and ESR1-e6>PCDH11X-induced growth remained sensitive to a CDK4/6 inhibitor, and a patient-derived xenograft (PDX) naturally expressing the ESR1-e6>YAP1 fusion was also responsive. Transcription...

Cancer Research, 2017

Estrogen receptor positive (ER+) breast cancer is treated with endocrine therapy but intrinsic re... more Estrogen receptor positive (ER+) breast cancer is treated with endocrine therapy but intrinsic resistance occurs in ~1/3 of patients and acquired resistance in ~1/5 of the remainder. While many resistance mechanisms have been explored, therapeutic strategies to overcome resistance in the clinical setting have seen mixed outcomes, and appear most effective in the acquired resistance setting. Understanding mechanisms of resistance and finding therapeutic strategies to target them, therefore, remain important challenges facing breast cancer researchers. In this study we systematically examine the role of DNA damage repair defects in inducing endocrine therapy resistance, a relatively understudied question of recent interest. We use in silico analysis of clinical datasets, in vitro experiments evaluating endocrine therapy resistance in response to DDR dysregulation in multiple breast cancer celllines, and in vivo validation using cellline xenograft and patient-derived xenograft models. ...

Cancer discovery, Oct 11, 2017

Significant endocrine therapy-resistant tumor proliferation is present in ≥20% of estrogen recept... more Significant endocrine therapy-resistant tumor proliferation is present in ≥20% of estrogen receptor positive (ER+) primary breast cancers and is associated with disease recurrence and death. Here, we uncover a link between intrinsic endocrine therapy resistance and dysregulation of the MutL mismatch repair complex (MLH1/3, PMS1/2), and demonstrate a direct role for MutL complex loss in resistance to all classes of endocrine therapy. We find that MutL deficiency in ER+ breast cancer abrogates Chk2-mediated inhibition of CDK4, a prerequisite for endocrine therapy responsiveness. Consequently, CDK4/6 inhibitors (CDK4/6i) remain effective in MutL-defective ER+ breast cancer cells. These observations are supported by data from a clinical trial where a CDK4/6i was found to strongly inhibit AI-resistant proliferation of MutL-defective tumors. These data suggest that diagnostic markers of MutL deficiency could be used to direct adjuvant CDK4/6i to a population of breast cancer patients who ...

International journal of biological sciences, 2016

Breast cancers exhibit high intertumoral heterogeneity in genetic alterations as well as histopat... more Breast cancers exhibit high intertumoral heterogeneity in genetic alterations as well as histopathological and other phenotypic characteristics. The contribution of the initiating oncogenic mutation to tumor phenotype remains controversial, largely due to the technical difficulties in delivering genetic alterations into well-defined subsets of mammary epithelial cells. To examine how different initiating oncogenes drive tumor phenotype, we somatically delivered two oncogenes (ErbB2, PyMT) into a narrow and distinct subset of the mouse mammary epithelium defined by the expression of the progenitor marker keratin 6a (Krt6a), and compared the phenotypes of the resulting mammary tumors. While PyMT-induced tumors were well-differentiated and displayed glandular and papillary features, ErbB2-induced tumors were poorly differentiated and exhibited epithelial-to-mesenchymal transition as well as β-catenin activation. These in vivo data demonstrate that the initiating oncogene plays a key ro...

eLife, 2013

While a first pregnancy before age 22 lowers breast cancer risk, a pregnancy after age 35 signifi... more While a first pregnancy before age 22 lowers breast cancer risk, a pregnancy after age 35 significantly increases life-long breast cancer risk. Pregnancy causes several changes to the normal breast that raise barriers to transformation, but how pregnancy can also increase cancer risk remains unclear. We show in mice that pregnancy has different effects on the few early lesions that have already developed in the otherwise normal breast—it causes apoptosis evasion and accelerated progression to cancer. The apoptosis evasion is due to the normally tightly controlled STAT5 signaling going astray—these precancerous cells activate STAT5 in response to pregnancy/lactation hormones and maintain STAT5 activation even during involution, thus preventing the apoptosis normally initiated by oncoprotein and involution. Short-term anti-STAT5 treatment of lactation-completed mice bearing early lesions eliminates the increased risk after a pregnancy. This chemoprevention strategy has important impli...

Proceedings of the National Academy of Sciences, 2010

Sirtuin 1 (SIRT1) is a class III histone deacetylase that deacetylates histone and nonhistone pro... more Sirtuin 1 (SIRT1) is a class III histone deacetylase that deacetylates histone and nonhistone proteins to regulate gene transcription and protein function. Because SIRT1 regulates very diverse responses such as apoptosis, insulin sensitivity, autophagy, differentiation, and stem cell pluripotency, it has been a challenge to reconcile how it orchestrates such pleiotropic effects. Here we show that SIRT1 serves as an important regulator of Wnt signaling. We demonstrate that SIRT1 loss of function leads to a significant decrease in the levels of all three Dishevelled (Dvl) proteins. Furthermore, we demonstrate that SIRT1 and Dvl proteins complex in vivo and that inhibition of SIRT1 leads to changes in gene expression of Wnt target genes. Finally, we demonstrate that Wnt-stimulated cell migration is inhibited by a SIRT1 inhibitor. Because the three mammalian Dvl proteins serve as key messengers for as many as 19 Wnt ligands, SIRT1-mediated regulation of Dvl proteins may explain the dive...

Molecular Endocrinology, 2013